Over the past decade, scanning confocal laser microscopy (SCLM) has revolutionized life science imaging. In scanning confocal laser microscopy, laser light is scanned across a specimen at a precisely chosen focal plane. Light reflected back from the specimen is collected, excluding light from all but the specifically-illuminated confocal plane. By excluding light reflected from all but the chosen image plane, glare is eliminated, producing crisp sectional images from full-thickness, unfixed tissues and cells. In addition, the reproducible spatial precision of the computer-driven scanning process permits the exact spatial registration of the individually-acquired sectional images, allowing the tomographic reconstruction of a three-dimensional image by overlay of the severally-acquired sectional images. Wiesendanger, Scanning Probe Microscopy and Spectroscopy: Methods and Applications, Cambridge Univ. Press (July 1995); Cullander, J. Investig. Dermatol. Symp. Proc. 3:166–171 (1998); Paddock, Proc. Soc. Exp. Biol. Med. 213:24–31 (1996); Ockleford, J. Pathol. 176:1–2 (1995); Laurent et al., Biol. Cell. 80:229–240 (1994).
The use of laser-excitable fluorescent dyes and proteins as ligand-specific probes has permitted scanning laser microscopy to be adapted beyond standard cell and tissue imaging to a wide variety of assays. Thus, laser scanning cytometers have proven particularly useful in fluorescence-based cytometric assays of cell cycle events. Juan et al., Methods Mol. Biol., 91: 67–75 (1996); Juan et al., Cell Biol. 2: 261–273 (1998); Juan et al., Cell Biol. 2: 341–350 (1998); Clatch et al., Cytometry 34: 36–38 (1998); Luther et al., Microscopy & Microanalysis, 3: 235–236 (1997).
Ashby et al., U.S. Pat. No. 5,549,588, describes scanning laser microscopic assay of “genome reporter matrices.” In these genome reporter matrices, each element of a spatially-addressable matrix contains cells in which expression of a common fluorescent reporter is driven from a distinct transcriptional regulatory element. The strength of the fluorescence signal acquired during scanning identifies the level of gene expression driven by each spatially-identifiable transcriptional regulatory element.
Scanning laser microscopy has also been adapted to scanning of nucleic acid microarrays built on silicon chips, Lashkari et al., Proc. Natl. Acad. Sci. USA 94: 13057–62 (1997); DeRisi et al., Science, 278:680–86 (1997); Wodicka et al., Nature Biotechnology, 15:1359–67 (1997); to measurement of ionic fluxes in cells, Schild, Cell. Calcium 19:281–296 (1996); Turner et al., J. Investig. Dermatol. Symp. Proc. 3:136–142 (1998); and to measurement of the subcellular distribution of various cellular components, Takubo et al., Haematologica 82:643–647 (1997).
Yet each of these applications of SCLM demands a specialized piece of computer-controlled optical equipment. There thus exists a need in the art for an inexpensive generic device that permits computer-driven confocal laser scanning of a microscopic sample.
The minimum mechanical requirements for such a device—laser, focusing and detection optics, precision scanning means, and computer interface—may all be found in standard optical disc readers or writers. Optical disc reader/writers, such as for CD and DVD, focus light from a solid state laser on a surface of a spinning disc and scan the disc to detect information that is encoded digitally in spatially-addressable patterns of submicron features.
Adaptation of optical discs and optical disc readers/writers to scanning microscopic applications would present marked advantages over existing approaches. Principal among these are availability and cost. The worldwide installed base of CD and DVD-ROM readers is estimated at present to be about 300 million units, and is expected within the next 5 years to rise to over 500 million units. Optical Publishing Industry Assessment, 9th ed. (Infotech, Inc., Woodstock, Vt.) (1998). The devices are inexpensive, reliable, and ubiquitous.
Other advantages of using optical discs for detection and characterization of microscopic structures are discussed in WO 96/09548 (Gordon), EP A 392475 (Idemitsu), EP A 417 305 (Idemitsu), EP A 504432 (Idemitsu), WO 98/28623 (Gamera), and WO 98/12559 (Demers), all of which are incorporated herein by reference. Further advantages are set forth in co-owned and copending U.S. patent applications Ser. No. 08/888.935, filed Jul. 7, 1997, Ser. No. 09/064,636, filed Apr. 21, 1998, Ser. No. 09/120,049 filed Jul. 21, 1998, and counterpart international applications published as WO 98/38510, Wo 98/38510 and WO 98/01533, the disclosures of which are incorporated herein by reference. There thus exists a need in the art for means to adapt optical disc readers to scanning laser microscopic applications.
Although optical disc readers possess the mechanical prerequisites for effective confocal laser microscopic scanning, operational requirements of existing disc readers present significant impediments to the successful detection and characterization of microscopic structures disposed upon the surface of an optical disc.
There are at least four basic operational requirements that must be satisfied for an optical drive correctly to read and decode the data present within an optical disc: the reader must focus correctly on the disc plane encoding the data, it must control the radial positioning of its optical pickup, it must control the tangential positioning of its optical pickup, and it must control the speed of disc rotation. The most common optical disc systems use elements of the optical medium itself to satisfy at least some of these requirements.
Thus, in a typical pressed CD, the disc substrate is impressed with a spiral track made up of a series of embossed pits, the signals from which are used by the optical disc reader to maintain proper focus and tracking. In CD-R, the data-encoding dye marks written by the user provide the requisite tracking features during subsequent reading. More generally, in each of the existing optical disc standards, the features used to encode data serve simultaneously to provide operational signals that the reader requires to control its operations. Although efficient, such standards make no provision for acquiring data from nonoperational features disposed upon the disk.
For example, because the tracking features are obligately embedded within the data layer of the disk, structures applied to the laser-proximal surface of the disc may interfere with detection of such operational features, and thus interfere with correct operation of the reader. Furthermore, such nonoperational structures may lie sufficiently outside the focal plane of the disc's operational features as to prevent their concurrent and discriminable detection by the reader's optical pickup.
One solution to this problem is to use nonstandard drives. One such proposed drive uses two optical pickups, one to detect tracking information, the other to detect surface structures, EP A 417 305 (Idemitsu). However, such modification moots a principal advantage of using optical disc readers for laser microscopic detection, which is the ecumenical distribution of such devices.
There thus exists a need in the art for optical discs that permit a standard optical disc reader/writer to acquire signals from nonoperational features of the disc, such as analyte-specific signal elements disposed thereon, concurrently and discriminably with signals generated by operational features of the disk, such as tracking attributes.